GSM7119414: HiCuT, E9.5, placenta, c57(m) x pwk(p); Mus musculus; OTHER
The HiCuT experiments were performed by Wuhan Frasergen Bioinformatics Co., Ltd. according to previous studies (7-9) with minor modifications. Briefly, placenta samples were cross-linked with 1% formaldehyde for 10 minutes at room temperature and quenched with 0.125M final concentration glycine for 5 minutes. Formaldehyde-fixed cells were subjected to additional cross-linking with 3 mM EGS, freshly prepared for 40 minutes and then quenched with 0.4 M glycine for 10 minutes at room temperature. Formaldehyde and ethylene glycol bis (succinimidylsuccinate) dual cross-linked cells were washed once with PBS supplemented with 0.05% BSA and were subsequently permeated with 0.05% digitonin for minutes at room temperature followed by 0.1% SDS at 65℃ for minutes. SDS were quenched by 1.8% Triton X-100 at 37℃ for minutes. Then chromatin DNA were digested by 50U MboI + 50U DdeI (NEB) at 37℃ for 2 hours, and fill-in with dNTP (Invitrogen) using 15U Klenow (NEB) at 37℃ for 1 hour. Spatial proximal DNA were ligated by 50U T4 DNA ligase (NEB) at 16℃ for 4 hours. Then cells were washed twice in Wash Buffer (20mM HEPES pH 7.5; 150mM NaCl; 0.5mM Spermidine; 1× Protease inhibitor cocktail) by gentle pipetting. Activated concanavalin A-coated bead-bound cells were resuspended in Dig-wash Buffer (Wash Buffer containing 0.05% Digitonin) containing 2mM EDTA and a 1:50 dilution of the primary antibody (H3K27ac antibody: CST, #8173). Primary antibody incubation was performed on a rotating platform at 4℃ overnight. Then the liquid was removed on the magnet stand. Secondary antibody (goat anti-rabbit IgG: Proteintech, B900210) was diluted 1:50 in Dig-Wash buffer and cells were incubated at room temperature for 1 hour. Cells were washed twice in Dig-Wash buffer to remove unbound antibodies. A 1:200 dilution of pG-Tn5 adapter complex (~0.04 μM) was prepared in Dig-300 Buffer (0.01% Digitonin, 20mM HEPES, pH 7.5, 300mM NaCl, 0.5mM Spermidine, 1× Protease inhibitor cocktail). Cells was incubated with pG-Tn5 at room temperature for 1 hour, then were washed twice in Dig-300 Buffer to remove unbound pG-Tn5 protein. Next, cells were resuspended in Tagmentation buffer (10mM MgCl2 in Dig-300 Buffer) and incubated at 37 °C for 1 hour. Tagmentation were stopped by incubating at 55 °C for 1hour containing 16mM EDTA, 0.1% SDS and 0.16 mg/mL Proteinase K. Then DNA was purified using phenol–chloroform–isoamyl alcohol and ethanol, and amplified by PCR reaction as follows: 3 min at 72 °C and 30 s at 98 °C followed by 16 cycles of 15 s at 98 °C and 30 s at 60 °C and 30 s at 72 °C, with a final extension at 72 °C for 3 min. Finally, the amplified DNA was purified using Ampure XP beads. Libraries were sequenced on Illumina NovaSeq platform.